THERMODYNAMICS AND THE DESIGN OF STRUCTURED PEPTIDES

热力学和结构肽的设计

• 批准号: 6636323
• 负责人: Niels Hjorth Andersen
• 金额: $ 17.71万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636323
• 关键词: calorimetry; chemical bond; circular dichroism; infrared spectrometry; interferometry; intermolecular interaction; mathematical model; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; peptide structure; protein engineering; protein folding; thermodynamics

The efficient folding of polypeptide sequences into stable structures is one of the most fascinating and fundamentally important biorecognition phenomena. Studies of the thermodynamics of this process provide insights into the factors that are required for avid binding of peptide drugs and hormones to their receptors. As a result, there is an increasing interest in the de novo design of protein-like scaffolds and the redesign of protein domains to be of the minimum size required for efficient self-assembly. The present proposal addresses peptide structuring requisites as a fold optimization and de novo design problem and also at a more fundamental level (the thermodynamics and rates of secondary structure formation and how these influence the rates and efficiency of protein folding, the quantitation of the deltaG increments due to specific hydrophobic and H-bonding interactions in miniprotein constructs). Some of the key experiments proposed should provide: a) the thermodynamic parameters for the formation of isolated alpha helices and beta hairpins and b) the relative importance of the hydrophobic effect for the stabilization of secondary structures. Some of the initial studies will be conducted in aqueous fluoroalcohol media that appear to accentuate the hydrophobic effect. However, the designed peptides proposed should allow the extension of these studies to strictly aqueous medium. The measurements of the rates of both alpha helix and beta hairpin formation will employ isotope-edited T-jump FT-IR. Stable alpha helices are promising units of scaffolding for designed folds; the helix/coil model developed at U.W. will be extended and reparameterized as a helix design tool and several key questions concerning intrinsic helical propensities and the stabilization increments associated with sidechain interactions will be addressed experimentally. Several novel miniprotein folds are proposed for study. One series consists of non-crosslinked eicosamers that fold cooperatively to produce a hydrophobic cage about a tryptophan ring. Mutants of this structure will be employed for the deltadeltaG measurements, as a model for developing strategies for the optimization of hydrophobic clusters, and as a test case for computer simulations of protein folding. A smaller effort will be directed at the construction and optimization of a betaalphaalpha miniprotein (a parallel beta sheet resulting from the association of beta strands at each end of an alpha helix). These constructs will mimic some features of the B1 domain of protein L but will have a left-handed crossover which has never been observed in nature. The studies described should provide insights and algorithms that will be useful in other peptide structure and ligand/receptor interface optimization efforts. The tryptophan cage fold, which will be examined extensively, is a unique intramolecular example of a binding motif that is common in intermolecular biorecognition phenomena. As a result, this project will provide strategies for designing more stable scaffolds of predictable structure for artificial enzymes and principles for the design of more potent biomolecules.

将多肽序列有效地折叠成稳定的结构是最引人入胜且根本重要的生物识别现象之一。 对此过程的热力学的研究提供了对肽药物和激素与受体狂热结合所必需的因素的见解。 结果，人们对蛋白质样支架的从头设计以及蛋白质结构域的重新设计的兴趣越来越大，这是有效自组装所需的最小尺寸。 本提案将肽结构必需品作为折叠优化和从头设计问题以及更基本的水平（二级结构形成的热力学和速率以及这些如何影响蛋白质折叠的速率和效率，由于特定的液生质和H键相互作用而导致的蛋白质折叠的速率和效率）。 提出的一些关键实验应提供：a）形成分离的α螺旋和β发夹的热力学参数以及b）疏水效应对二级结构稳定的相对重要性。 一些初步研究将在氟酒精培养基中进行，这些培养基似乎会突显疏水作用。 但是，提出的设计肽应允许将这些研究扩展到严格的水性培养基。 Alpha螺旋和β发夹形成速率的测量将采用同位素编辑的T-JUMP FT-IR。稳定的Alpha螺旋是设计褶皱的有前途的脚手架。 U.W.开发的螺旋/线圈模型将扩展并重新聚集为螺旋设计工具，并将与Sidechain相互作用相关的固有螺旋倾斜度以及与Sidechain相互作用相关的稳定增量进行实验解决。 提出了几种新型的微蛋白折叠。 一个系列由非交联的eicosamers组成，它们合作地折叠以产生疏水笼，上面有色氨酸环。 该结构的突变体将用于三角洲测量值，作为开发优化疏水簇的策略的模型，以及用于蛋白质折叠的计算机模拟的测试用例。 较小的努力将针对βAlphaalpha微蛋白的构建和优化（由Alpha螺旋的每一端的β链相关的平行β片）。 这些构建体将模仿蛋白质L的B1域的某些特征，但具有左手的跨界，这在自然界中从未观察到。所描述的研究应提供洞察力和算法，这些算法和算法将在其他肽结构以及配体/受体界面优化工作中有用。 色氨酸笼子折叠将进行广泛检查，是结合基序的独特分子内示例，在分子间生物识别现象中很常见。 结果，该项目将为设计更稳定的人工酶的可预测结构的脚手架和设计更有效的生物分子的原理提供策略。